A universal stress protein upregulated by hypoxia has a role in Burkholderia cenocepacia intramacrophage survival: Implications for chronic infection in cystic fibrosis

Abstract Universal stress proteins (USPs) are ubiquitously expressed in bacteria, archaea, and eukaryotes and play a lead role in adaptation to environmental conditions. They enable adaptation of bacterial pathogens to the conditions encountered in the human niche, including hypoxia, oxidative stress, osmotic stress, nutrient deficiency, or acid stress, thereby facilitating colonization. We previously reported that all six USP proteins encoded within a low‐oxygen activated (lxa) locus in Burkholderia cenocepacia showed increased abundance during chronic colonization of the cystic fibrosis (CF) lung. However, the role of USPs in chronic cystic fibrosis infection is not well understood. Structural modeling identified surface arginines on one lxa‐encoded USP, USP76, which suggested it mediated interactions with heparan sulfate. Using mutants derived from the B. cenocepacia strain, K56‐2, we show that USP76 is involved in host cell attachment. Pretreatment of lung epithelial cells with heparanase reduced the binding of the wild‐type and complement strains but not the Δusp76 mutant strain, indicating that USP76 is directly or indirectly involved in receptor recognition on the surface of epithelial cells. We also show that USP76 is required for growth and survival in many conditions associated with the CF lung, including acidic conditions and oxidative stress. Moreover, USP76 also has a role in survival in macrophages isolated from people with CF. Overall, while further elucidation of the exact mechanism(s) is required, we can conclude that USP76, which is upregulated during chronic infection, is involved in bacterial survival within CF macrophages, a hallmark of Burkholderia infection.


| INTRODUCTION
Many opportunistic bacterial pathogens must adapt as they transition from their natural environment to the host to survive within the host and establish an infection. Changes in temperature, pH, osmolarity, and oxygen availability are among the stresses that bacteria must overcome when they colonize the mammalian niche. Universal stress proteins (USPs) are extensively expressed throughout nature from bacteria to archaea and eukaryotes in response to a wide variety of environmental conditions although they have not yet been identified in humans . This breadth of evolutionary range illustrates the importance of USPs in the resilience of organisms to survive in stressful environments, and while their role in bacterial cells is predominantly involved in the adaptive response to the changing environment, their contributions to bacterial pathogenesis cover a range of different mechanisms . In pathogens such as Mycobacterium tuberculosis and Pseudomonas aeruginosa, USPs are upregulated within macrophages and/or in response to low oxygen conditions, supporting survival under conditions of stress Hingley-Wilson et al., 2010;Schreiber et al., 2006), while an Acinetobacter baumannii USP has a protective role against low pH and contributes to pathogenesis (Elhosseiny et al., 2015). Recently, a USP was identified as a key determinant for the robustness of the nosocomial pathogen Enterococcus faecium during starvation (de Maat et al., 2020).
USPs were first identified in Escherichia coli K-12 following exposure of cells to a variety of stresses, including heat shock, carbon and nitrogen starvation, and ultraviolet radiation (Nyström & Neidhardt, 1996).
Expression of UspA in E. coli was independent of the stringent stress response transcriptional activators RelA/SpoT, RpoH, KatF, OmpR, AppY, Lrp, PhoB, and H-NS (Nyström & Neidhardt, 1992). E. coli uspA gene deletion mutants were defective in survival over prolonged periods of growth under stress conditions such as induced peroxide stress and osmotic shock (Nyström & Neidhardt, 1994). M. tuberculosis USP Rv2623 contributes negatively to virulence by regulating its growth in the transition to latency (Drumm et al., 2009). There are six usp genes in the E. coli genome, while there are 10 usp genes encoded in the M. tuberculosis genome .
Burkholderia cepacia complex (Bcc) is a group of Gram-negative bacteria that naturally occurs in soil and causes chronic opportunistic life-threatening infections in people with cystic fibrosis (CF) and immunocompromised patients. It can also colonize pharmaceutical plants and contaminate pharmaceutical products and disinfectants (Vanlaere et al., 2009). It is highly antimicrobial resistant and once a chronic infection has been established, eradication is rare. Its capacity to colonize diverse and harsh niches is exemplary, and consequently, elucidation of its mechanisms of adaptation is essential Sass et al. (2013) identified a 50 gene locus that was dramatically upregulated under low oxygen conditions and designated the low oxygen activated (lxa) locus (Sass et al., 2013). We subsequently showed that 19 proteins encoded on the lxa locus showed increased abundance in late infection isolates from chronically colonized CF patients relative to early isolates (Cullen et al., 2018). These late chronic infection isolates also showed increased attachment to CF lung epithelial cells relative to their respective early isolates (Cullen et al., 2017). Importantly, all six USPs encoded on the lxa locus showed increased protein abundance with the time of colonization (Cullen et al., 2018) and five of which were regulated by the DNA mimic protein, Bnr1 (Dennehy et al., 2022).
Among these lxa-encoded usp genes, BCAM0276 encodes a UspA family stress protein which consistently showed increased abundance in the later isolates from two chronically colonized patients and was associated with increased gene expression (Cullen et al., 2018).
Previously, this USP (USP76) was reported to be upregulated almost 60-fold in B. cenocepacia strain J2315 in response to low oxygen (Sass et al., 2013), and up to 40-fold in a comparative transcriptomic study of B. contaminans isolates from CF patients (Nunvar et al., 2016). There are 11 USPs in total encoded on the B. cenocepacia genome (Winsor et al., 2008) CF lung disease is characterized by recurrent bacterial infections, exacerbations, and excessive pulmonary inflammation (Elizur et al., 2008;Roesch et al., 2018). Limited oxygen is another hallmark of the CF lung due to rapid oxygen consumption by microorganisms; neutrophils; impaired ventilation; and mucous plugging (Montgomery et al., 2017).
These selective pressures in CF airways drive adaptation in colonizing bacteria, enabling them to overcome the CF lung microenvironments during infection. Moreover, pathogens such as Bcc can survive phagocytosis and even replicate within macrophages, contributing to chronic infection (Martin & Mohr, 2000;Rosales-Reyes et al., 2012). To examine the role of USPs in this process, we compared two USPs encoded on the lxa-locus, USP76, and USP92, which have comparable predicted sizes (

| Bacterial strains and growth conditions
The strains and plasmids used in this study are listed in the Appendix (Table A1). Bacteria were routinely grown at 37°C in lysogeny broth (LB) with orbital shaking (200 rpm) unless otherwise stated.
Antibiotics, when required, were added to reach final concentrations as follows: 50 μg/ml trimethoprim for E. coli and 100 μg/ml for B. cenocepacia, and 40 μg/ml kanamycin for E. coli.

| Mammalian cell culture
CF epithelial cells, CFBE41o − which are homozygous for the ΔF508 mutation of the CFTR gene were routinely cultured in collagen/ fibronectin-coated flasks as previously described (Cullen et al., 2017).

| Construction of Δusp mutants and complementation
Targeted gene deletions of BCAM0276 or BCAM0292 in the B. cenocepacia strain K56-2 were performed as described (Flannagan et al., 2008) and named as Δusp_76 and Δusp_92, respectively. The amplicons used to construct the mutagenic plasmid were cloned into pGPI-SceI-2 digested with EcoRI and NheI, using triparental mating, followed by biparental mating to introduce the I-SceI endonuclease.
The screening was performed on the resulting colonies from bi-parental mating to determine if successful gene deletion had taken place. Trimethoprim-sensitive colonies were screened for gene deletion using the US forward DS reverse primers (Appendix 1, Table A2) and mutants were sent for sequencing (Eurofins Genomics Ltd) to confirm sequence deletion and stocks in glycerol prepared.
To complement B. cenocepacia K56-2ΔBCAM0276, wild-type BCAM0276 was amplified from B. cenocepacia K56-2 with the complementation primer pairs (Table A2), digested with the restriction enzymes NdeI and XbaI and ligated into similarly digested pMH447 (Hamad et al., 2012). The complementation plasmid was introduced into the mutant by conjugation. Once transferred into the target mutant strain the complementation vector integrates into the genome at aminoglycoside efflux genes (BCAL1674-BCAL1675), due to sequence homology between the vector and target genome (Hamad et al., 2010). As before, pDAI-Sce-I was then introduced resulting in the replacement of BCAL1674-1675 by BCAM0276.
The complementation of the BCAM0276 gene was confirmed by polymerase chain reaction (PCR) and by phenotype analysis.
Following genetic manipulation of B. cenocepacia K56-2 strain during gene deletion and subsequent complementation, the presence of the plasmid pC3 was confirmed using three sets of primers designed for genes repA, oriC, and dopC. The pC3 plasmid is a nonessential replicon of B. cenocepacia and encodes for a number of virulence factors (Agnoli et al., 2014), which can be lost during genetic manipulation. PCR was performed on confirmed mutants and complements using primers 3001/3002, 3003/3004, and 3005/3006.

| Bacterial attachment to human CF epithelial cells
CFBE41o − cells were seeded in wells of a 24-well plate at a density of 4 × 10 5 cells/well in antibiotic-free medium and incubated overnight.
The cells were then washed three times with phosphate-buffered saline (PBS) and the mid-logarithmic phase bacterial cultures (OD 600nm 0.6-0.8) were resuspended in MEM and added to each well at a concentration of 2 × 10 7 CFU/well (MOI 50:1).

Low pH
Overnight cultures were diluted 1:100 LB at either pH 4.5 or standard pH~7.5 and 300 μl aliquots added in duplicate to the wells of a 96-well round-bottomed plate. The plates were incubated at 37°C, with orbital shaking and OD 600nm measured every 15 min for 20 h. Cell viability in low pH was also determined by inoculating a 100 ml flask of LB at pH 4.5 with 10 ml of an overnight culture and incubating at 37°C, 170 rpm, and plating hourly samples as described previously.

Oxidative stress
The effects of oxidative stress on the B. cenocepacia strains were assessed firstly by exposing the strains to a series of concentrations

High osmolarity
The effect of high osmolarity was determined in each B. cenocepacia strain by adding 300 μl of a range of concentrations of NaCl (0%-5% w/v) or sucrose (0%-50%) to rows of a 96-well plate. Overnight cultures were added to each well (3 μl) in duplicate and the plates were incubated in a Biotek Synergy H1 multiplate reader at 37°C for 24 h with OD 600nm measured every 15 min.

Heat stress at 42°C
The effect of heat on each B. cenocepacia strain was determined by transferring 10 ml of overnight cultures of each strain into 100 ml of prewarmed (42°C) LB and incubated at 42°C, 200 rpm. Hourly samples were diluted in Ringer's solution, plated, and enumerated after 48 h.

| Bacterial uptake and survival in U937 macrophage cells
The internalization of B. cenocepacia strains by U937 macrophages was determined as previously described (McKeon et al., 2010).
Briefly, overnight cultures of each strain were transferred to fresh LB and incubated until mid-logarithmic phase (OD 600nm of 0.6-0.8) and diluted to an MOI of 5:1 (2.5 × 10 6 CFU/ml) in RPMI medium (McKeon et al., 2010). U937 cells were seeded at 5 × 10 5 cells/ml in a 24-well plate, differentiated with PMA, washed twice with PBS, and 1 ml of each bacterial culture applied in duplicate. The CFU applied was confirmed by serial dilution of aliquots in Ringer's and plating in duplicate. The 24-well plates were centrifuged at 1100g for 5 min, and incubated at 37°C, 5% CO 2 for 2 h, before washing with PBS.
Extracellular bacteria were killed with amikacin/ceftazidime (1 mg/ml each) for 2 h at 37°C, 5% CO 2 . Wells were then washed five times with sterile PBS before cells were lysed with 0.25% Triton X-100 for 15 min and scraped, diluted, and plated as outlined above. The final wash was also plated to verify that all extracellular bacteria were killed. The % uptake was calculated as the intracellular CFU/ml at a 2-h point relative to the CFU/ml applied to the cells at time zero. To examine intracellular survival of B. cenocepacia in U937 macrophages, bacteria were incubated with U937 as described above and after 2 h of incubation with each strain, the wells were washed PBS before the addition of RPMI amikacin/ceftazidime (1 mg/ml each) to each well and incubation for a further 2 h at 37°C, 5% CO 2. The wells were subsequently washed twice with PBS and fresh antibiotics replaced and incubated at 37°C, 5% CO 2 for up to 24 h. Cells were then washed, lysed, diluted, and plated as described above and CFU determined after 48 h.

| Isolation of human peripheral mononuclear cells (PBMC) and differentiation
Age and gender-matched adults with CF with no history of Bcc Assessment of antibiotic resistance, mucoidy, motility, and biofilm formation was performed as described in Appendix 3.

| Structural features of USP76 and USP92
The BCAM0276 and BCAM0292 genes are two of six USP-encoding genes located on the lxa locus (Sass et al., 2013). We previously reported that USP76 and USP92, showed up to 6-and 16-fold increased abundance, respectively, from early to late infection in two sets of sequential isolates and were selected for further investigation (Cullen et al., 2018). Moreover, we showed that the abundance of these was reduced by 1.  (Figure 1), consistent with their 41.8% sequence identity and 58% homology (Altschul, 1997) which are above the expected threshold for protein similarity (Rost, 1999). Each monomer is composed of an open-twisted, five-strand parallel β-sheet, sandwiched by two α-helices on each side of the sheet (α1-α4 in

| The Δusp76 mutant has a reduced attachment to host cells
We previously showed that the attachment of sequential B.
cenocepacia isolates to CF epithelial cells, CFBE41o − , increased over the time of chronic infection (Cullen et al., 2017). Given the observed increase in BCAM0276 gene expression and a corresponding increase in USP76 protein abundance (Cullen et al., 2018)  It has been shown that the lxa locus, including the BCAM0276 gene, was dramatically upregulated in response to low oxygen (Sass et al., 2013) therefore it was important to understand if USP76 was involved in survival in response to low oxygen. The K56-2 strain was unable to grow in oxygen environments lower than 6% oxygen and survival was considerably impaired at 6% oxygen ( Figure 5a). When exposed to 6% oxygen in a controlled hypoxia chamber, no Δusp76 mutant cells survived to Day 8, indicating that this gene is critical to extended survival under low oxygen.
Complementation of the gene in the Δusp76_usp76 strain restored survival to near WT levels.
The CF lung is also characterized by constant inflammation and the production of reactive oxygen species due to persistent bacterial infections. The impact of the oxidative environment on the survival of the Δusp76 mutant was examined using hydrogen peroxide. Growth

| USP76 is required for growth and survival at acidic pH
Several bacterial USPs support survival under acidic conditions. The growth of the Δusp76 mutant in LB at low pH (pH 4.5) was reduced (p < 0.003) over 20 h relative to WT when normalized for growth in LB at physiological pH (~pH 7.4, Figure 5c). Growth of the Δusp76_usp76 complemented strain was comparable to WT levels.
When the strains were plated to determine the viable CFU remaining after exposure to acidic pH over a range of time points, it was apparent that the viability of the K56-2 strain following growth in acidic conditions was also dependent on USP76 expression ( Figure 5d, p = 0.0092). The Δusp76_usp76 complement strain showed an ability to recover, reaching WT levels within 5 h of exposure to acidic pH.
3.6 | USP76 has no impact on antibiotic susceptibility or response to osmotic stress The CF lung and the airway surface liquid also represent high salt environments with 1% w/v NaCl concentrations reported for the ASL of people with CF compared to 0.7% in healthy controls (Grandjean Lapierre et al., 2017). This creates osmotic stress on any pathogen colonizing the CF lung. The growth of the Δusp76 mutant was comparable to that of the WT strain over a range of salt concentrations (0%-5% NaCl, p = 0.72) indicating that USP76 does not play a role in the salt tolerance of B. cenocepacia (Appendix 2, Figure A4).

| Deletion of usp76 does not affect mucoidy, motility, or biofilm formation
Alterations in mucoid phenotype have been associated with chronically infecting Bcc isolates (Zlosnik et al., 2008), however, deletion of the usp76 gene did not impact exopolysaccharides (EPS) production when grown on Yeast extract mannitol (YEM) agar plates in either normoxic or hypoxic conditions (Appendix 1, Table A3; Appendix 2, Figure A5). Motility was comparable between the WT and the Δusp76 mutant strain (Appendix 2, Figure A6). Nonstatistically significant differences were observed for the motility phenotype when analyzed by ANOVA, swimming p = 0.9720, swarming p = 0.2044, and twitching p = 0.4226. In addition, the ability of B. cenocepacia to form biofilms was not affected by the presence of the usp76 gene (Appendix 2, Figure A7) under normal oxygen conditions or hypoxic conditions.

| DISCUSSION
A hallmark of the genus Burkholderia is its remarkable ability to survive and thrive in a range of diverse environments, ranging from the soil, aquatic niches, and disinfectants to the human host. Its ability to adapt to changing environments contributes to its success as a human pathogen. USPs are ubiquitous proteins that play a wide and UspD mutants were more susceptible to oxidative and superoxide stress (Nachin et al., 2005;Nyström & Neidhardt, 1994).
Listeria monocytogenes UspA mutants exposed to acidic stress were also previously found to have reduced cellular growth (Seifart Gomes   et  cenocepacia in macrophages, particularly given that USP76 was also protective in oxidative stress and involved in host cell attachment. The impaired survival of the Δusp76 mutant in U937 macrophage cells and, crucially, our finding that survival was significantly impaired in 80% of the CF patient-derived macrophage samples confirms that USP76 confers a clear survival advantage to B. cenocepacia inside macrophage cells. People with CF can have impaired macrophage function, which can lead to altered phagocytosis and the killing of bacteria (Leveque et al., 2017), consequently, the role of USP76 in uptake and survival in CF patient-derived macrophages is particularly noteworthy. The finding that impaired survival of the Δusp76 mutant was not evident in all CF patient-derived macrophages samples, is not unexpected given the heterogeneity of human macrophages (Turton et al., 2021) and it is likely that other host factors, including infection status, also play a role (Leveque et al., 2017). For example, the levels of IL-8 secretion by CF macrophages depend on the patient's lung function and the severity of the patient's lung disease (Leveque et al., 2017;Simonin-Le Jeune et al., 2013). It should be noted that while PBMCs were collected from both males and females with CF, we were unable to successfully culture sufficient macrophages from any female samples. The BCAM0276 gene was previously shown to be upregulated when B. cenocepacia strain K56-2 was internalized in murine macrophages   (Nachin et al., 2005). Moreover, E. coli UspA and UspD were required for protection against oxidative stress while UspC and UspE were not. As with USP76, E. coli USPs have also been linked to cell adhesion when mutants were assessed by yeast agglutination, with mutants of UspC and UspE enhancing cellular attachment, UspF and UspG mutants reducing attachment (Nachin et al., 2005). The identification of surface arginine residues on USP76 and not USP92 suggested that these were likely to play a key role in host A question remains as to how a protein such as USP76, which is predicted to be cytosolic, mediates host cell attachment at the cell surface and can also mediate survival in stressful conditions such as low oxygen, acidic environments, or within macrophages. This multiplicity of roles has been observed with E. coli USPs previously, with UspF and UspG playing roles in both oxidative stress and host cell attachment (Nachin et al., 2005). In the case of these E. coli USPs, their host cell attachment role is associated with fimbriation. We cannot exclude a similar indirect effect in Usp76-mediated attachment to host cells. However, it should be noted that there is increasing evidence of bacterial proteins with moonlighting functions, that show intracellular enzymatic functions and additional functions as receptors or adhesins to host proteins or tissues (Henderson & Martin, 2011;Wang et al., 2014). A key focus is establishing how USP76, which is predicted to be a cytosolic protein, is present on the bacterial cell surface. It is possible that consistent with many other predicted cytosolic proteins including many E. coli proteins (Monteiro et al., 2021), USP76 has an extracytoplasmic location. Recent work on O157 E. coli strains showed that 16, 14, and 31 predicted cytosolic proteins were located in the proteosurfaceome, proteovesiculome, and exoproteome respectively (Monteiro et al., 2021).
OMV release by Vibrio cholerae in response to stress plays a comparable role in surface adaptation in vivo and colonization (Zingl et al., 2020), but this hypothesis and the potential release of USPs in outer membrane vesicles (OMVs) in response to stress will need to be further evaluated in B. cenocepacia. There have been over 300 moonlighting proteins identified to date (Chen et al., 2018), and research studies to elucidate this multifunctionality at multiple locations are yet to come.
In summary, USPs are critical for an environmental bacterium such as Burkholderia to cope with the breadth of stressful conditions that it is exposed to in soil. It is now clear that USPs also confer substantial advantages on opportunistic pathogens adapting to the niche environment of the CF lung and in particular, that USP76 is a major contributor to intramacrophage survival. Furthermore, the

CONFLICT OF INTEREST
None declared.

DATA AVAILABILITY STATEMENT
All data are provided in full in the results section of this paper.  Figure A1 APPENDIX 3

Assessment of mucoid phenotype
Exopolysaccharide (EPS) production was assessed to determine the mucoid phenotype of the mutant strains of Bcc using Yeast extract mannitol (YEM) (2 g/L Yeast extract, 20 g/L D-mannitol, 15 g/L Agar no.2) plates (Lagatolla et al., 2002). Overnight cultures of each strain were streaked onto YEM plates in a quadrant streak manner and incubated at 37°C for 5 days in normal oxygen conditions or low oxygen conditions using a controlled hypoxic F I G U R E A1 Growth of wild-type, Δusp76 and Δusp92 mutant strains over 24 h incubation in LB at 37°C with shaking. Data shown represent the mean OD 600nm ± standard deviation. chamber (COY). EPS production and mucoid phenotype were scored as follows: The scoring of mucoid phenotype was as follows: (−) bacterial colonies on YEM plates appeared dry with a matte appearance and showed no evidence of EPS production, (+) bacterial colonies predominately non-mucoid with some evidence of EPS production in the confluent area of growth on the YEM plate, (++) mucoid phenotypes with EPS production on both the confluent area of growth and streaked individual colonies which appeared flat on the YEM plates, (+++) excessive EPS production with a poor distinction between confluent area and streaked area, streaks appeared raised due to EPS production; a score of (+++d) was similar to (+++) but the EPS production resulted in drips of EPS on the lid of the Petri dish when inverted (Zlosnik et al., 2008).

Determination of motility
Motility assays were performed on each mutant strain to determine the effect of gene deletion on the ability of swimming, swarming, and twitching (Cullen et al., 2015). F I G U R E A4 Endpoint normalized OD 600nm of WT K56-2 and Δusp76 following a 24 h incubation with concentrations ranging from 0% to 5% W/V NaCl. Normalized endpoint absorbance values measured at 24 h incubation of WT K56-2 and Δusp76 at 37°C in various concentrations of NaCl ranging from 0% to 5% w/v. Values displayed have been normalized to 0% W/V NaCl (LB only with culture) and represent the average of 3 independent occasions. Statistical analysis determined by ANOVA (p = 0.72). ANOVA, analysis of variance; LB, lysogeny broth; WT, wild-type.
F I G U R E A5 Representative images of the mucoid phenotype of WT and Δusp76 strains in normal oxygen conditions and at 6% O 2 . EPS production of WT (a) and Δusp (b) incubated on YEM agar for 5 days at 37°C in either normal oxygen conditions (a, b) or 6% O 2 (c, d). Images represent mucoid phenotype on 1 of 3 independent occasions. EPS, exopolysaccharides; YEM, Yeast extract mannitol.  10% v/v acetic acid for 2 min. The excess stain was then washed off with a solution of 40% methanol and 10% acetic acid.
Twitching was determined by taking the average of two diameter measurements of the zone motility. Strains were scored as follows: nonmotile (diameter ≤5 mm), motile (diameter >5 mm and ≤30 mm) or highly motile (diameter of >30 mm).

Assessment of biofilm formation
Overnight cultures (10 ml) of each strain were transferred to 100 ml of fresh LB and incubated at 37°C for 4-5 h with agitation at 200 rpm.
OD 600nm was measured and cells were diluted to~1.6 × 10 6 CFU/ml in F I G U R E A7 Determination of biofilm formation of WT and Δusp76 mutant over a period of 72 h in normoxic and hypoxic conditions. Average biofilm formation for the WT and the Δusp76 mutant strain as determined by measuring OD 590nm of resolubilized, crystal violet stained biofilms in normal oxygen (a) and hypoxic (6% O 2 ) (b) conditions measured every 24 h for 72 h. Data represent the mean OD 590nm from three independent occasions. Error bars represent the standard error of each mean. WT, wild-type.